POSTER: High Throughput Proteomic Applications Using anti-DYKDDDDK Magnetic Agarose

Speaker
  • R&D Staff Scientist, Protein and Cell Analysis, Thermo Fisher Scientific
    Biography
      Betsy Benton completed her PhD and postdoctoral research at the University of Wisconsin-Madison in the fields of biochemistry and human oncology. She joined Thermo Fisher Scientific in 2000, where she has developed numerous products in the areas of protein purification (with both magnetic beads and resin supports), protein sample preparation, and protein detection.

    Abstract

    Affinity purification, immunoprecipitation, and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high-capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti-DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against a DYKDDDDK tag covalently attached to high-binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen™ ExpiCHO™ Expression System, and by a HeLa in vitro translation (cell-free) system. Biological activity was demonstrated for ExpiCHO-expressed DYKDDDDK–Tumor Necrosis Factor alpha (TNFα) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB) p65. In addition, successful co-immunoprecipitation of DYKDDDDK-tagged BAD with 14-3-3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor and mass spectrometry. In summary, anti-DYKDDDDK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications.

    Learning Objectives:

    • Learn how to automate protein applications using cell-based and cell-free systems with magnetic beads

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